Project Summary Urothelial carcinoma of the bladder (i.e. bladder cancer) is the most common primary tumor of the urothelial tract. Bladder cancer recurrence and metastases remain common and are significant contributors to patient morbidity and mortality. Despite the high prevalence of recurrence and metastasis, no new treatment options have been introduced for advanced disease in the past three decades. Many patients who initially respond to standard of care treatment exhibit refractory disease within a few years. Furthermore, patients with metastatic disease at the time of diagnosis often exhibit resistance to standard of care therapeutic treatment. These cases underscore the urgent need for new therapies. One potential target for new therapy is fibroblast growth factor receptor 3 (FGFR3), which is frequently mutated in bladder cancer. In vitro studies with FGFR3 inhibitors showed promising results, however, the in vivo responses were more modest. Currently, it is unknown what modulates response to FGFR3 inhibition in vivo, but I hypothesize that the microenvironment is contributing to resistance mechanisms in bladder cancer. The goal of this proposal is to better understand the roles that the tumor microenvironment plays in resistance of bladder cancers to Fibroblast Growth Factor Receptor 3 (FGFR3) inhibition. The overall hypothesis is that constituents of the tumor microenvironment confer resistance to FGFR3 inhibition and promote metastasis in bladder cancer, and that response may depend on cellular differentiation state. This hypothesis will be tested by employing MicroEnvironment MicroArrays (MEMA), which consists of robotically printed growth pads made up of combinations of functional extracellular matrix (ECM) components, growth factors and cytokines found in different local and metastatic microenvironments, allowing for systematic assessment of microenvironment effects on cellular phenotypes in a rational reductionist manner. Based on preliminary data, the specific aims of this proposal are as follows: (1) Determine the role that the microenvironment plays in resistance to FGFR3 inhibition in bladder cancer, and (2) Investigate the role that the microenvironment plays in expression of differentiation state markers in response to FGFR3 inhibition in bladder cancer. Results from this proposal have the potential to provide new information that will enable us to devise novel targeted approaches aimed at anticipating or overcoming resistance to FGFR3 inhibition and preventing metastases in this disease. Thus, this project has the potential to greatly impact bladder cancer patients' quality of life and overall survival.